The present invention relates to an animal model useful for testing potential therapeutic agents for the treatment of neurodegenerative disorders, in particular disorders associated with the presence of Lewy pathology.
Lewy pathology is a defining hallmark of degenerating neurons and/or glial cells in post mortem brain tissue of patients with neurodegenerative disorders including idiopathic Parkinson""s Disease (PD), dementia with Lewy bodies (DLB), a Lewy Body variant of Alzheimer""s Disease (LBVAD) and multiple system atrophies (MSA). Lewy-type changes seem central and may contribute mechanistically to dysfunction and degeneration of neurons and/or glial cells in these diseases. The characteristic appearance of Lewy pathology is known to the skilled artisan and described, e.g. in Spillantini et al., Proc. Natl. Acad. Sci. USA 95:6469-6473 (1998). Lewy pathology in neurons includes the aberrant distribution pattern, e.g. in soma and dendrites, and the finding of aggregates of the presynaptic protein xcex1-synuclein in neurons as compared to a predominantly axonal and presynaptic localisation in normal cells. Two mutations (A53T or A30P) in the xcex1-synuclein gene are linked to early-onset familial PD with Lewy pathology.
The lack of an experimental animal model showing neuropathological changes as observed in PD, DLB, LBVAD and MSA and reflecting underlying pathological mechanisms is a major obstacle that hampers significant advance in both basic research and drug development.
It has now surprisingly been found that features of Lewy pathology are evident in the brain of transgenic or somatic recombinant animals expressing an exogenous xcex1-synuclein gene under the control of nervous tissue specific regulatory sequences. The animals provide a first and novel model which can be used to test neuroprotective treatments for diseases involving xcex1-synucleinopathy.
Accordingly in a first aspect the invention provides transgenic or somatic recombinant non-human animals which exhibit xcex1-synucleinopathy. More particularly the invention provides transgenic or somatic recombinant non-human animals expressing exogenous xcex1-synuclein. Preferably they express exogenous xcex1-synuclein under the control of a nervous tissue specific regulatory sequence. More preferably the transgenic or somatic recombinant non-human animals overexpress exogenous xcex1-synuclein, e.g. express exogenous xcex1-synuclein under the control of a regulatory sequence selected from the group comprising a Thy-1 gene-regulatory sequence or a Tyrosine Hydroxylase (TH) gene-regulatory sequence.
In a further aspect the invention provides a recombinant DNA construct comprising a poly-nucleotide encoding an xcex1-synuclein polypeptide functionally linked to a nervous tissue specific regulatory sequence, e.g. a Thy-1- or a TH-regulatory sequence.
In a still further aspect a transgenic cell expressing exogenous xcex1-synuclein is provided, in particular a transgenic cell comprising a recombinant DNA construct comprising a poly-nucleotide encoding an exogenous (xcex1-synuclein polypeptide functionally linked to a nervous tissue specific regulatory sequence, and expressing said exogenous xcex1-synuclein.
Transgenic or somatic recombinant non-human animals include animals into which a suitable construct has been introduced, and, in the case of transgenic animals, progeny of such animals still retaining said construct. Examples for useful animal lines include any animal line normally kept as laboratory animals, e.g. mouse and rat lines. Very useful mouse lines are the C57BL/6 line and B6CF1 line.
xcex1-synucleinopathy in the animals shows several striking features of Lewy pathology as it presents itself in post mortem brain tissue of PD, LBVAD, DLB and MSA patients. Like in diseased human PD, LBVAD, DLB and MSA brains, subsets of neurons in the animals show xcex1-synuclein-stained perikarya and Lewy-like neurites. A small subset of these cells may also stain for ubiquitin.
xcex1-synucleinopathy may be analysed applying a panel of immuno- and histochemical techniques routinely used to assess Lewy pathology in human brain and well known in the art. The level of xcex1-synuclein mRNA expression may be analysed e.g. by RNA blotting, S1 nuclease protection techniques and/or RT/PCR (reverse transcription and polymerase chain reaction) technology, the expression pattern of the exogenous gene in the brain may be determined by in situ hybridization, and detection of xcex1-synuclein protein in the brain may be effected using immunoblotting (western blot analysis) and/or immunohistochemical staining techniques, and the effects of the expression may be studied by histology and immunohistology, as well as by using state-of-the-art high resolution protein technologies (e.g. proteomics) and/or high-resolution RNA expression profiling (gene chip technologies).
Typically observed heterogeneous changes involved in xcex1-synucleinopathy include sausage-like enlargements of proximal and distal neuritic segments, thick or fine thread-like inclusions, as well as beaded or spindle-shaped neurites. For example some of the most prominently involved cell groups and/or brain regions in the Thy-1-xcex1-synuclein transgenic or somatic recombinant non-human animals include the nucleus centralis oralis pontis, the nucleus vestibularis lateralis, the deep cerebellar nuclei, the deep aspects of the tectal plate, and motor nuclei in the spinal cord. In TH-xcex1-synuclein transgenic or somatic recombinant non-human animals, the potentially affected and involved cell groups include mainly the catecholaminergic neurons, including those located in the substantia nigra (that are also affected in patients with PD), and that have been shown to expess a TH- or a TH-driven transgene. Involved cell groups may further include those that have been shown to express TH transiently, e.g. in mouse, the Purkinje cells in cerebellum during postnatal development days P21-28, and/or cells in other brain regions.
In affected brain areas of the transgenic or somatic recombinant non-human animals, there is astrocytic gliosis and microglial activation. Spinal roots immunostain for xcex1-synuclein and axonal degeneration is apparent with nerve fibers showing breakdown and segmentation of their myelin sheaths into elipsoids. Skeletal muscle contains atrophic angular fibers indicating neurogenic muscular atrophy and they show loss of neuromuscular synapses. In agreement, the transgenic or somatic recombinant non-human animals show a progressive impairment of limb and motor function. Specific tests known in the art provide easy and also non-invasive read-outs for limb and motor function in animals.
xcex1-synuclein sequences include wildtype xcex1-synuclein, e.g. as disclosed in Maroteaux and Scheller, Brain Res. Mol. Brain Res. 11:335- (1991); Polymeropoulos et al., Science 276:2045-2047 (1997); Hong et al., Neuroreport 9:1239-1243 (1998); Genbank accession numbers L08850 (human xcex1-synuclein); AF007758 (rat xcex1-synuclein); and mutated (xcex1-synuclein, e.g. an xcex1-synuclein linked to early onset familiar PD, e.g. A53T, as disclosed in WO 98/59,050, and/or A30P, as disclosed in Krueger et al., Nature Genetics 18:106-108 (1998).
The transgenic or somatic recombinant non-human animals may be generated according to well established methods for introduction of a recombinant DNA construct allowing germ-line or somatic insertion including viral or non-viral vector-mediated gene transfer into fertilized eggs, zygotes or early embryos and/or a specific tissue (such as brain) in the adult animal, e.g. by gene transfer into embryonic stem cells, retroviral infection of early embryos or pronuclear microinjection. Further manipulation of resulting fertilized eggs, zygotes or early embryos and breeding of resulting transgenic founder animals follows established routes of breeding transgenic animals.
Recombinant DNA constructs useful in the present invention may be prepared according to procedures known in the art. For example a nervous tissue specific regulatory sequence may be identified and isolated starting from, e.g. routinely screening e.g. bacterial artificial chromosome (BAC) genomic DNA banks. A nervous tissue specific regulatory sequence is meant to be a regulatory sequence which is specifically active in nervous tissue, including, e.g. a regulatory sequence directing expression in neurons, in macroglial cells, e.g, astrocytes or oligodendrocytes, or in microglial cells. An example for a nervous tissue specific regulatory sequence are Thy-1 gene sequences; and an example for a Thy-1 regulatory sequence is a fragment obtainable from an approximately 8.1 kb mouse genomic Eco RI DNA fragment [Vidal et al. EMBO J. 9:833-840 (1990), Ingraham et al., J. Immunol. 136:1482-1489 (1986)], and comprises mouse Thy-1 promoter sequences, exon1, part of exon 2 and exon 4 and sequences 3xe2x80x2 of the last coding-exon, e.g. as illustrated in SEQ ID NO: 11. Another example for a nervous tissue specific regulatory sequence is a modified version of these Thy-1 regulatory sequences, in which an internal SstI restriction fragment in the intron is replaced by enhancer sequences derived from the immunoglobulin heavy chain locus, as described e.g. in Texido et al. [J. Immunol. 153:3028-3042 (1994)]. Still another example for a nervous tissue specific regulatory sequence are TH-gene sequences, e.g. those comprising e.g. 9 kb of the 5xe2x80x2 regulatory sequences in e.g. the rat tyrosine hydroxylase gene (Min et al., Mol. Brain Res. 27: 281-289 (1994)] and as e.g. illustrated in SEQ ID NO: 12. Expression of xcex1-synucleins in astrocytes may be directed using state of the art expression cassettes containing e.g. sequences of the Glial Fibrillary Acidic Protein Gene (GFAP) [Balcare and Cowen, Nucl. Acids Res. 13:5527-5543 (1985); Mucke et al., The New Biologist 3:465-474 (1991); Mucke and Rockenstein, Transgene 1:3-9 (1993); Brenner et al., J Neuroscience 14:1030-1037 (1994); Toggas et al., Nature 367:188-193 (1994); Mohajeri et al., Eur.J. Neuroscience 8:1085-1097 (1996)]. Expression in microglial cells can be achieved using state of the art regulatory sequences derived from the human Fc gamma RI gene [Heijnen and van de Winkel, J. Hematotherapy 4:351-356 (1995); Heijnen et al., J. Clin. Invest. 97:331-338 (1996)].
By e.g. modification of sequences comprising a nervous tissue specific regulatory sequence and using state-of-the-art technology [e.g. as described by Zhang et al., Nature Genetics 20:123-128 (1998); Muyrers et al., Nucleic Acid Res. 27:1555-1557 (1999)] for inserting the xcex1-synuclein encoding sequences in the desired location of the genomic sequences a re-combinant DNA construct is obtainable which may then be used in the preparation of transgenic or somatic recombinant non-human animals.
Transgenic cells expressing exogenous xcex1-synuclein may be prepared by any technique known in the art, for example the recombinant DNA construct may be introduced by direct DNA microinjection, DNA transfection, viral or non-viral vectors, or the cells may be obtained from transgenic or somatic recombinant non-human animals, and cultured in vitro.
Models based on cells and animals of the invention may be used for example to identify and assess the efficacy of potential therapeutic agents in neurodegenerative diseases, particularly in diseases where xcex1-synuclein is involved and/or Lewy-type pathology appears, more particularly in PD, DLB, LBVAD and MSA. In particular such models may be used in screening or characterization assays for detecting agents likely to modulate xcex1-synuclein related, derived, and/or evoked pathology. The animals of the invention may be used for testing estrogens or estrogen modulators for their therapeutic potential in preventing or treating diseases with xcex1-synucleinopathy.
Accordingly in a further aspect the invention comprises a method for testing a potential therapeutic agent for a specified condition, in particular a neurodegenerative disease, preferably PD, DLB, LBVAD or MSE, wherein a cell of the invention is used as target cell. More particularly it comprises such a method, wherein the agent is administered to a transgenic or somatic recombinant non-human animal of the invention. Moreover the invention comprises a screening or characterization assay consisting in or including such a method, as well as a screening assay kit comprising cells of the invention.
Methods for screening potential therapeutic agents using cell lines or animals are well known in the art. The cells and animals of the present invention may be used in analogous manner.
The recombinant cells may for example be incubated with the potential therapeutic agent and with antibodies recognizing xcex1-synuclein. In methods where transgenic or somatic recombinant non-human animals themselves are used, the effects of the potential therapeutic agent may be determined by carrying out various investigations on the animals after sacrifice. Also after administration of the potential therapeutic agent, transgenic or somatic recombinant non-human animals may undergo specific testing in order to monitor, e.g. motor functions.
In a further aspect the present invention is directed to a novel modulator of xcex1-synuclein distribution pattern and aggregation identified by a screening assay comprising incubating a cell expressing exogenous xcex1-synuclein under the control of a nervous tissue specific regulatory sequence with the potential modulator and measuring and assessing changes.
The present invention also embodies an animal model for the identification of an indicator, which presence, absence or disregulation are hallmarks of predisposition, onset, progression, halt and/or reversal of the disease. For example, analysis of e.g. blood samples of transgenic or somatic recombinant non-human animals expressing an exogenous xcex1-synuclein and at various stages, i.e. ages and/or stages of a manifesting Lewy-like disease process may be used to identify such indicators. The transgenic or somatic recombinant non-human animals provide a novel means to identify in single xcex1-synuclein protein species pathological changes associated with xcex1-synucleinopathy. This approach is also useful to detect changes in proteins other than xcex1-synuclein. Overall, it illustrates the potential of the animal models to discover novel protein species that are specifically associated with the development of xcex1-synucleinopathy. The novel protein species offer potential as novel drug targets for therapy, as reagents to develop novel e.g. antibodies for diagnostic purposes, and/or as surrogate markers. Furthermore, the type of modifications that distinguishes these proteins from their normal counterparts provide links to pathways that can be exploited therapeutically in the context of xcex1-synucleinopathy. The transgenic or somatic recombinant non-human animals provide a novel means to identify pathological changes associated with xcex1-synucleinopathy and in single protein species, which potential can be exploited e.g. as targets for therapy or as surrogate markers. Functional changes in the animals can be recorded using some behavioural paradigms. For example, by recording altered locomoter activity in the transgenic animals as compared to their non-transgenic littermates, following an injection of low doses of cocaine (e.g. 10 mg/kg).
In accordance with the foregoing the present invention thus provides
(1) A transgenic or somatic recombinant non-human animal, e.g. mammal, e.g. a rodent, e:g. a rat or a mouse, which exhibits xcex1-synucleinopathy, e.g. a transgenic or somatic recombinant non-human animal expressing exogenous xcex1-synuclein, e.g. a transgenic or somatic recombinant non-human animal expressing exogenous xcex1-synuclein under the control of a nervous tissue specific regulatory sequence, e.g. expressing exogenous xcex1-synuclein under the control of a regulatory sequence selected from the group comprising a Thy-1 regulatory sequence and a TH regulatory sequence.
(2) A transgenic or somatic recombinant non-human animal, e.g. mammal, e.g. a rodent, e.g. a rat or a mouse, comprising and expressing an exogenous polynucleotide encoding an xcex1-synuclein polypeptide, e.g. wildtype xcex1-synuclein or a mutated xcex1-synuclein, e.g. an xcex1-synuclein linked to early onset familiar PD, e.g. A53T and/or A30P, functionally linked to a nervous tissue specific regulatory sequence, e.g. a Thy-1- or a TH-regulatory sequence.
(3) A recombinant DNA construct comprising a polynucleotide encoding an xcex1-synuclein polypeptide, e.g. wildtype xcex1-synuclein or a mutated xcex1-synuclein, e.g. an xcex1-synuclein linked to early onset familiar PD, e.g. A53T and/or A30P, functionally linked to a nervous tissue specific regulatory sequence, e.g. a Thy-1- or a TH-regulatory sequence.
(4) A transgenic cell expressing exogenous xcex1-synuclein, e.g. comprising and expressing a recombinant DNA construct as under (3).
(5) A method of producing a transgenic or somatic recombinant non-human animal as under (1) or (2) wherein said animal is generated by introducing a recombinant DNA construct as under (3) into the genome of germ-line or somatic cells, e.g. by viral or non-viral vector-mediated gene transfer into fertilized eggs, zygotes or early embryos and/or a specific tissue in the unborn, e.g. the embryo, or born, e.g. young or adult, animal, and breeding of resulting transgenic founder animals or maintaining resulting transgenic animals.
(6) A method for testing a potential therapeutic agent for modulating Lewy pathology wherein the agent is administered to a transgenic or somatic recombinant animal as under (1) or (2) or is contacted with a cell according to (4) and xcex1-synuclein distribution pattern and aggregation is determined.
(7) A method for screening a compound or a combination of compounds for the ability to prevent, revert and/or stop cells from undergoing change to Lewy pathology comprising contacting a cell as under (4) or an animal as under (1) or (2) with the compound or combination of compounds and observing xcex1-synuclein distribution pattern and aggregation.
(8) A method for screening a compound or a combination of compounds, e.g. an estrogen or an estrogen modulator, for its potential to prevent or treat a disease with xcex1-synucleinopathy, comprising contacting an animal as under (1) or (2) with the compound or combination of compounds and comparing the results obtained in a test for motor deficits, e.g. Rotating Rod test, with treated animals vs. untreated animals, less motor deficits being indicative for a therapeutic potential.
(9) A screening or characterization assay consisting in or including a method as under (7) or (8).
(10) A screening assay kit comprising cells as under (4).
(11) A compound for use in the treatment of a neurodegenerative disease associated with the presence of Lewy pathology which has been identified by a method according to (6), (7) or (8) or by using an assay or an assay kit according to (9) or (10).
(12) A composition for preventing, reverting and/or stopping neural cells from undergoing change to Lewy pathology, which composition is effective to modulate xcex1-synuclein distribution pattern and aggregation in neural cells.
(13) A method for treating a patient suffering from a neural condition comprising administering to the patient a pharmaceutically effective amount of a composition effective to modulate xcex1-synuclein distribution pattern and aggregation in neural cells.
(14) A method for the identification of an endogenous indicator of predisposition, onset, progression, halt and/or reversal of human diseases associated with Lewy pathology comprising analysis of changes detectable in animals as under (1) or (2) or cells as under (4) and/or the causes thereof.
The following examples illustrate the invention without limitation.